α-glucan phosphorylase (hereinafter, also referred to as GP) is an enzyme utilized in, for example, synthesis of glucose-1-phosphate (hereinafter, also referred to as G-1-P), and glucan synthesis. G-1-P is utilized, for example, as a medical antibacterial agent, an anti-tumor agent (as a platinum complex), a drug for treating heart disease (as an amine salt), or a substrate for glucan synthesis. GP is widely distributed in plants, for example in tubers such as potatoes; animals, for example in rabbit muscle; and microorganisms such as yeast.
Among the above, plant-derived GP is useful because it generally has the ability to synthesize glucans having a high molecular weight.
Various GPs can be used to produce G-1-P or glucans, inter alia, potato-derived GP is used in many cases because a relatively large amount of the enzyme is easily obtained.
In industrial production of G-1-P or a glucan using GP, it is necessary to essentially remove other enzyme activity derived from contamination of GP, particularly, phosphatase activity and amylase activity Escherichia coli and Bacillus subtilis are desirable hosts to express a GP gene when producing large amounts of GP. However, as shown in FIG. 4 and FIG. 5, Escherichia coli has amylase activity and phosphatase activity, and Bacillus subtilis has amylase activity. However, as shown in FIGS. 4 and 5, enzymes expressed by these hosts cannot be inactivated by heat treatment at 55° C., but can be almost completely inactivated by heat treatment at 60° C. Therefore, a plant-derived GP having heat resistance whereby it's activity is not lost, even after heat treatment at 60° C., has been desired.
For reference, specific numerical values of amylase activity and phosphatase activity in cell extracts from various bacteria (Escherichia coli TG-1 strain, Escherichia coli BL21 strain, and Bacillus subtilis ANA-1 strain) before and after heating are shown in the following Table 1.
TABLE 1PhosphataseAmylaseactivityactivity(%)(%)TG-1BL21TG-1BL21ANA-1Before100100100100100heating50° C.99.198.621.628.633.855° C.60.974.59.19.719.860° C.2.93.10.403.065° C.2.52.00.902.4
However, a plant-derived GP which can synthesize high molecular weight glucans, and has thermostability, particularly, GP which can maintain sufficient activity at high temperatures (e.g. 60° C. to 75° C.), is not known. Regarding GP derived from organisms other than plants, GP having high thermostability, GP expressed by extreme thermophilic bacteria (Thermus aquaticus, Thermococcus litoralis, Aquifex aeolicus and the like) has been reported. However, since such the above GP is derived from organisms other than plants, it is unable to synthesize high molecular weight glucans, and is thus less useful.
GPs are classified into two groups based on homology between amino acid sequences, (see Non-Patent Document 1). GP having 30% or more identity to potato-derived GP is classified as being a group A GP, and a GP having less than 30% identity to potato-derived GP and having 30% or more identity to GP of Thermus aquaticus is classified a being a group B GP.
A glucan produced using GP derived from Thermus belonging to a group B has a considerably lower molecular weight when compared with a glucan produced using potato-derived GP which is classified as a group A GP. For this reason, there is the problem that high molecular weight glucans cannot be obtained using GP derived from Thermus. 
In order to solve these problems, a plant-derived GP which is advantageous for industrial utilization, and has high thermostability, is required.
Theoretical methods for making a general enzyme more thermostable, such as proline theory and amino acid substitution based on enzyme steric structure information have been tried, but have not necessarily succeeded. For this reason, methods based upon random mutation, or methods using a combination of random mutation and theoretical methodology is currently being carried out. However, in any of these methods, every protein must be characterized by trial and error.
Regarding enzymes other than GP, it has been reported that, once the position of a particular amino acid(s) involved in improving the thermostability of an enzyme is, determined, an enzyme can be made thermostable by substitution of the specified amino acid residue(s) with other amino acid residues (for example see Non-Patent Documents 3 to 5).
An example of GP having improved thermostability has been reported with regard to Escherichia coli maltodextrin phosphorylase (see Non-Patent Document 2). In this document, thermostable Escherichia coli maltodextrin phosphorylase is disclosed. Maltodextrin phosphorylase is one type of GP. In this GP having improved thermostability, asparagine at position 133 is substituted with alanine. This asparagine at position 133 is present at an active site, and is a binding site for pyridoxal 5′-phosphate which is a coenzyme essential in the enzymatic reaction. In this GP having improved thermostability, thermostability is improved by about 15° C., and the optimal reaction temperature is elevated from about 45° C. to about 60° C., and the GP is denatured at about 67° C., as compared with natural GP. However, this Escherichia coli GP, similar to Thermus-derived GP, does not have the ability to synthesize high molecular weight glucans h. Further, the enzyme activity at optimal temperatures for the GP having improved thermostability described in this document, is lower than the enzyme activity at an optimal temperature of natural GP. That is, due to mutation, the ability to synthesize a glucan thereof, is lowered. For this reason, this document teaches that substitution at position 133 is not preferable, at least from the viewpoint of glucan synthesizing ability.
Usually, an enzyme protein is unstable, and is sensitive to physical factors such as pH, temperature etc, as well as proteases, and thus may be easily degraded. Among enzymes, there are also enzymes which become more unstable, and therefore easily degraded, at high degrees of purification. For this reason, enzymes must be prepared at as low as possible temperatures, and must be prepared before every use. Degradation of an enzyme can be suppressed by freezing and storing. However, proteins are degraded upon thawing in some cases, and handling is therefore difficult when an enzyme is stored frozen and subsequently thawed. Generally, when an enzyme is degraded, the steric structure changes, and the nature of the enzyme with regard to optimal pH, pH stability, reaction rate, substrate affinity, and the like, similarly changes. Occasionally the enzyme activity is lowered, or inactivated. As such, degradation of an enzyme protein greatly influences enzyme reaction. For this reason, for industries that utilizing enzymes, it is desirable to use enzymes that have excellent stability as far as possible.
It has been known that natural potato type L GP is also easily degraded and, even when purified GP is refrigerated and stored, it gradually degrades from the point of purification. When degradation of a GP protein can be suppressed, it becomes possible to prepare a large amount of GP and store it long term, thus increasing production efficiency, which is a significant advantage in terms of both storage and use of an enzyme. For this reason, it is also preferable to provide GP which can be stored long term, without degradation.
(Non-Patent Document 1)
Takeshi Takaha, et el., “Structure and Properties of Thermus aqaticus α-Glucan Phosphorylase Expressed in Escherichia coli”, J. Appl. Glycosi., 2001, Vol. 48, No. 1, pp. 71-78
(Non-Patent Document 2)
Richard Grieβler, et al., “Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli”, Biochem. J., 2000, 346, pp. 255-263
(Non-Patent Document 3)
Martin Lehmann and Markus Wyss, “Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution”, Current Opinion in Biotechnology, 2001, 12, pp. 371-375
(Non-Patent Document 4)
M. Lehmann, et al., “The consensus concept for thermostability engineering of proteins”, Biochemica Biophysica Acta, 2000, 1543, pp. 408-415
(Non-Patent Document 5)
Junichi Miyazaki, et al., “Ancestral Residues Stabilizing 3-Isopropylmalate Dehydrogenase of an Extreme Thermophile: Experimental Evidence Supporting the Thermophilic Common Ancestor Hypothesis”, J. Biochem, 2001, 129, pp. 777-782